Accumulation of omega-7 fatty acids in plant seeds

ABSTRACT

Compositions and methods include genetically encoding and expressing a novel Δ 9 -18:0-ACP desaturase in plant cells. In some embodiments, nucleic acid molecules encode the novel Δ 9 -18:0-ACP desaturase. In other embodiments, amino acid sequences have Δ 9 -18:0-ACP desaturase activity. Methods can involve expression of Δ 9 -18:0-ACP desaturase in plant cells, plant materials, and whole plants for the purpose of increasing the amount of unusual fatty acids in whole plants, plant seeds, and plant materials, for example, seeds.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/358,318, filed Jun. 24, 2010, the disclosure ofwhich is hereby incorporated herein in its entirety by this reference.

FIELD OF THE INVENTION

In particular embodiments, the invention relates to a novel mutantRicinus Δ ⁹-18:0-ACP desaturase, designated Com25, that functions as aΔ⁹-16:0-ACP desaturase. Another embodiment relates to methods ofmetabolic engineering to manipulate metabolic branch points in plants,for example, to redirect carbon into ω-7 fatty acids. In certainembodiments, the invention relates to methods for expressing Com25 aspart of a metabolic engineering strategy, such that carbon is redirectedinto ω-7 fatty acids in plant seeds.

BACKGROUND

It has been estimated that there may be upwards of 1,000 fatty acidstructures in nature. Millar et al., (2000) Trends Plant Sci5(3):95-101. Many of these fatty acids are synthesized by derivatizationof the fatty acids by an array of variants of archetypal desaturases.The first of these variant desaturases to be isolated was the Ricinusoleate hydroxylase from castor endosperm, the enzyme responsible forricinoleic acid synthesis. van de Loo et al., (1995) Proc. Natl. Acad.Sci. USA 92(15):6743-6747. This was followed by the genes encoding theVernonia linoleate epoxidase and the Crepis oleate actylenase. Lee etal., (1998) Science 280(5365):915-18. The isolation of these genes ledto the notion that their heterologous expression in oil crop plantscould facilitate the accumulation of the corresponding unusual fattyacids. Broun et al., (1997) Plant Journal 13:201-10. However, theresulting unusual fatty acid accumulation was invariably lower than thatfound in the natural source plant from which the gene was isolated.Napier, J. A. (2007) Annu. Rev. Plant Biol. 58:295-319.

The specific activity profiles of variant desaturase enzymes that havebeen isolated from tissues that accumulate unusual fatty acids areconsistent with a role of producing the corresponding unusual fattyacids. However, they exhibit very poor specific activities compared withall stearoyl-ACP desaturases reported to date and have provedineffective in producing altered fatty acid phenotypes whenheterologously expressed. Cahoon et al., (1994) Prog. Lipid Res.33:155-63. For instance, seed-specific expression of the castorhydroxylase under the control of a strong seed-specific promoter in themodel plant Arabidopsis resulted in the accumulation of only about 17%of ricinoleic acid, far short of the about 90% found in castor seed.Broun and Somerville (1997) Plant Physiol. 113:933-42. Similarlydisappointing results have been reported for epoxy and acetylenic fattyacids which have been reported to accumulate to 15 and 25% respectivelyupon heterologous expression of the epoxygenase and acetylenase inArabidopsis. Lee et al., (1998) Science 280(5365):915-18. In addition toshowing poor activities, variant desaturases tended to form insolubleaggregates when purified. Low stability and poor catalytic rates areproperties shared by many newly evolved enzymes that arise as geneduplication events in which selection for stability and/or turnover isreleased, while mutations accumulate that finally result in alterationof function. Govindarajan and Goldstein (1998) Proc. Natl. Acad. Sci.USA 95:5545-49; Goldstein (2001) in Protein Folding, Evolution andDesign (Broglia, R. A., Shakhnovich, E. I., and Tiana, G., eds) CXLIVVols., I.O.S. Press, Amsterdam.

Many potential explanations for low levels of target fatty acidaccumulation have been advanced. Napier, J. A. (2007) Annu. Rev. PlantBiol. 58:295-319. Evidence suggests specialized enzymes may play a keyrole in the incorporation of the unusual fatty acid intotriacylglycerols. For instance, the accumulation of laurate intransgenic Brassica napus seeds increased from 50% to 60% upon thecoexpression of a coconut lysophosphatidic acid acyltransferase alongwith the California bay medium chain thioesterase. Knutzon et al.,(1999) Plant Physiol. 120(3):739-46. Recently, coexpression of thecastor type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2)along with the castor hydroxylase increased the accumulation ofricinoleic acid from about 17% to about 30%. Burgal et al., (2008) PlantBiotechnol. J. 6(8):819-31.

Accumulating high levels of unusual fatty acids in transgenic plantsequivalent to those found in naturally occurring species has yet to bereported. As unusual fatty acids are highly desirable in a variety ofindustries and applications, there is a need for better expression ofunusual fatty acids in transgenic plants designed for their production.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein are nucleotide sequences encoding a novel variantdesaturase, designated Com25, and the amino acid sequence thereof.

Also disclosed are methods of expressing Com25 in a plant cell, to takeadvantage of the Com25 enzyme's enhanced desaturase activity, relativeto the WT castor Δ⁹-18:0 desaturase, such that the percent compositionof unusual fatty acids in plant seeds is increased. In some embodiments,the methods include expressing Com25 in Arabidopsis. In certainembodiments, the unusual fatty acids increased in plant seeds are ω-7fatty acids. In these embodiments, the ω-7 fatty acids may be 16:1Δ⁹and/or 18:14¹¹.

Methods are also provided for expressing Com25 in a plant cell, whereinthe plant cell is impaired in plastidial and extraplastidial fatty acidelongation, such that the percent composition of unusual fatty acids inplant seeds is increased. In some embodiments, the methods includeexpressing Com25 in Arabidopsis. In certain embodiments, the unusualfatty acids increased in plant seeds are ω-7 fatty acids. In theseembodiments, the ω-7 fatty acids may be 16:1Δ⁹ and/or 18:1Δ¹¹.

Further methods are provided for expressing Com25 in a plant cellwherein KASII is inhibited in the plant cell, such that the percentcomposition of unusual fatty acids in plant seeds is increased. In someembodiments, the methods include expressing Com25 in Arabidopsis. Incertain embodiments, the unusual fatty acids increased in plant seedsare ω-7 fatty acids. In these embodiments, the ω-7 fatty acids may be16:1Δ⁹ and/or 18:1Δ¹¹.

Methods are also provided for expressing Com25 in a plant cell whereinKASII and plastidial and extraplastidial fatty acid elongation areinhibited in the plant cell, such that the percent composition ofunusual fatty acids in plant seeds is increased. In some embodiments,the methods include expressing Com25 in Arabidopsis. In certainembodiments, the unusual fatty acids increased in plant seeds are ω-7fatty acids. In these embodiments, the ω-7 fatty acids may be 16:1Δ⁹and/or 18:1Δ¹¹.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic of fatty acid synthesis and modification inthe plastid and endoplasmic reticulum of Arabidopsis. Reactions mediatedby 16:0 desaturases are indicated 1: Δ⁹-16:0-ACP desaturase; 2:extraplastidial Δ⁹-16:0-ACP desaturase. ω-7 FA, i.e., 16:1 Δ⁹ and 18:1Δ¹¹ are boxed.

FIG. 2 displays a representative gas chromatographic separation of FAMEsupon expression of Com25 in various backgrounds of Arabidopsis. Panels Aand B, WT; C and D, fab1; E and F, fab1/fae1. Panels A, C and E,untransformed; B, D and F, transformed with Phas:Com25. FAME peaks areindicated: 16:0 (1), 16:1Δ⁹ (2), 16:2 (3), 18:0 (4), 18:1 Δ⁹ (5),18:1Δ¹¹ (6), 18:2 (7), 20:0 (8), 20:1Δ¹¹ (6), 18:2 (7), 20:0 (8),20:1Δ¹¹ (9), 18:3+20:1Δ¹³ (10), and 22:1 (11).

FIG. 3 shows the relationship between 16:0 in host seeds versus ω-7accumulation (as mol percent).

FIG. 4 displays a representative gas chromatographic separation of FAMEsupon expression of Com25 in various backgrounds or Arabidopsis. Panel A:best fab1/fae1, Phas:Com25, Fab1-HPAS, AnΔ9DS, LnΔ9DS transformant line;Panel B: Doxantha seed. Peak designations are as described in FIG. 2.

FIG. 5. is a schematic arrangement of DNA elements in particularconstruct embodiments of the invention.

DETAILED DESCRIPTION I. Overview of Several Embodiments

Disclosed herein are nucleotide acid molecules encoding a Δ⁹ desaturaseenzyme comprising a nucleotide sequence at least 60% identical to SEQ IDNO:1. The nucleic acid molecules may further comprise a gene regulatoryelement. In some embodiments, the gene regulatory element may be aphaseolin promoter.

Also disclosed are Δ⁹ desaturase enzymes comprising an amino acidsequence at least 80% identical to SEQ ID NO:2. Δ⁹ desaturase enzymes ofthe present invention wherein the amino acid sequence is at least 80%identical to SEQ ID NO:2 may further comprise a serine at the positionanalogous to position 114 in SEQ ID NO:2; an arginine at the positionanalogous to position 117 in SEQ ID NO:2; a cysteine at the positionanalogous to position 118 in SEQ ID NO:2, a leucine at the positionanalogous to position 179 in SEQ ID NO:2; and/or a threonine at theposition analogous to position 188 in SEQ ID NO:2.

Nucleic acid molecules and Δ⁹ desaturase enzymes of the presentinvention may be expressed in plant materials, cells, tissues, or wholeplants, to increase the amount of unusual fatty acids in the plantmaterial, cells, tissues, or whole plants, relative to the amountobserved in a wild type plant of the same species. Alternativeembodiments of the invention include methods for increasing the amountof unusual fatty acids in the plant material, cells, tissues, or wholeplants comprising transforming plant material, cells, tissues, or wholeplants with the nucleic acid molecule of SEQ ID NO:1, such that theamount of unusual fatty acids in said plant material, cells, tissues, orwhole plants is increased.

In preferred embodiments, the plant material, cells, tissues, or wholeplants that are transformed by the disclosed methods further compriseone or more means for increasing levels of 16:0-ACP in the plantmaterial, cells, tissues, or whole plants. In certain embodiments, themeans for increasing the levels of 16:0-ACP in the plant material,cells, tissues, or whole plants may be: expression of an extraplastidialdesaturase; suppression of KASII, for example by introducing a mutationin the fab1 gene; and/or decreasing the elongation of 16:0 fatty acids,for example by introducing a mutation in the fae1 gene.

Disclosed methods herein may be performed, for example, on plants, orplant materials derived from plants, of the genus Arabidopsis. Aparticular embodiment is drawn to methods for creating or regenerating agenetically engineered plant comprising increased amounts of unusualfatty acids in the plant compared to a wild type plant of the samespecies, comprising transforming plant material with nucleic acidmolecule of SEQ ID NO:1; and culturing the transformed plant material toobtain a plant. Plants, plant materials, plant cells, and seeds obtainedby the aforementioned methods are also disclosed.

II. Abbreviations

-   -   x:yΔ^(z) fatty acid containing x carbons and y double bonds in        position z counting from the carboxyl end    -   ACP acyl carrier protein    -   COA coenzyme A    -   KASHII β-ketoacyl-ACP synthase II    -   FA fatty acids    -   FAS fatty acid synthesis    -   FAME fatty acid methyl ester    -   WT wild type

III. Terms

Fatty acid: As used herein, the term “fatty acid” refers to long chainaliphatic acids (alkanoic acids) of varying chain lengths, from aboutC12 to C22, although both longer and shorter chain-length acids areknown. The structure of a fatty acid is represented by the notation,x:yΔ^(z), where “x” is the total number of carbon (C) atoms in theparticular fatty acid, and “y” is the number of double bonds in thecarbon chain in the position “z,” as counted from the carboxyl end ofthe acid.

Unusual fatty acid: For the purposes of the present invention, unusualfatty acids are those whose synthesis in natural systems is initiated bymodification of an intermediate of FAS by a variant desaturase enzyme.

Metabolic pathway: The term, “metabolic pathway,” refers to a series ofchemical reactions occurring within a cell, catalyzed by enzymes, toachieve either the formation of a metabolic product, or the initiationof another metabolic pathway. A metabolic pathway may involve several ormany steps, and may compete with a different metabolic pathway forspecific reaction substrates. Similarly, the product of one metabolicpathway may be a substrate for yet another metabolic pathway.

Metabolic engineering: For the purposes of the present invention,“metabolic engineering” refers to the rational design of strategies toalter one or more metabolic pathways in a cell, such that thestep-by-step modification of an initial substance into a product havingthe exact chemical structure desired is achieved within the overallscheme of the total metabolic pathways operative in the cell.

Desaturase: As used herein, the term “desaturase” refers to apolypeptide that can desaturate (i.e., introduce a double bond) in oneor more fatty acids to produce a fatty acid or precursor of interest.Plant-soluble fatty acid desaturase enzymes introduce a double bondregiospecifically into a saturated acyl-ACP substrate. The reactioninvolves activation of molecular oxygen by a two-electron reduced diironcenter coordinated by a four-helix bundle that forms the core of thedesaturase architecture. Of particular interest herein are Δ⁹desaturases.

The Δ⁹-18:0¹-ACP desaturase is required by all plants for themaintenance of membrane fluidity. While this enzyme primarilydesaturates stearoyl-ACP, it is also active to a minor extent withpalmitoyl-ACP.

Variant desaturase: As used herein, the term “variant desaturase”encompasses those desaturases that exhibit specific activity profilesconsistent with a role in producing unusual fatty acids. A variantdesaturase may be isolated from an organism, or engineered via adirected evolution program.

Progeny plant: For the purposes of the present invention, “progenyplant,” refers to any plant, or plant material obtained therefrom, thatmay be obtained by plant breeding methods. Plant breeding methods arewell-known in the art, and include natural breeding, artificialbreeding, selective breeding involving DNA molecular marker analysis,transgenics, and commercial breeding.

Plant material: As used herein, the term “plant material” refers to anycell or tissue obtained from a plant.

Nucleic acid molecule: A polymeric form of nucleotides, which caninclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide, or a modified form of either type ofnucleotide. A “nucleic acid molecule” as used herein is synonymous with“nucleic acid” and “polynucleotide.” The term includes single- anddouble-stranded forms of DNA. A nucleic acid molecule can include eitheror both naturally occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules can be modified chemically or biochemically, orcan contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of ordinary skill in the art. Suchmodification include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications, such as uncharged linkages (for example,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.), charged linkages (for example, phosphorothioates,phosphorodithioates, etc.), pendent moieties (for example, peptides),intercalators (for example, acridine, psoralen, etc.), chelators,alkylators, and modified linkages (for example, alpha anomeric nucleicacids, etc.). The term “nucleic acid molecule” also includes anytopological conformation, including single-stranded, double-stranded,partially duplexed, triplexed, hairpinned, circular and padlockedconformations.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence is ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.When recombinantly produced, operably linked nucleic acid sequences aregenerally contiguous and, where necessary to join two protein-codingregions, in the same reading frame. However, nucleic acids need not becontiguous to be operably linked.

Regulatory element: As used herein, the term “regulatory element” refersto a nucleic acid molecule having gene regulatory activity; i.e., onethat has the ability to affect the transcription or translation of anoperably linked transcribable nucleic acid molecule. Regulatory elementssuch as promoters, leaders, introns, and transcription terminationregions are non-coding nucleic acid molecules having gene regulatoryactivity which play an integral part in the overall expression of genesin living cells. Isolated regulatory elements that function in plantsare therefore useful for modifying plant phenotypes through thetechniques of molecular engineering. By “regulatory element,” it isintended a series of nucleotides that determines if, when, and at whatlevel a particular gene is expressed. The regulatory DNA sequencesspecifically interact with regulatory proteins or other proteins.

As used herein, the term “gene regulatory activity” refers to a nucleicacid molecule capable of affecting transcription or translation of anoperably linked nucleic acid molecule. An isolated nucleic acid moleculehaving gene regulatory activity may provide temporal or spatialexpression or modulate levels and rates of expression of the operablylinked nucleic acid molecule. An isolated nucleic acid molecule havinggene regulatory activity may comprise a promoter, intron, leader, or 3′transcriptional termination region.

Promoters: As used herein, the term “promoter” refers to a nucleic acidmolecule that is involved in recognition and binding of RNA polymeraseII or other proteins such as transcription factors (trans-acting proteinfactors that regulate transcription) to initiate transcription of anoperably linked gene. Promoters may themselves contain sub-elements suchas cis-elements or enhancer domains that effect the transcription ofoperably linked genes. A “plant promoter” is a native or non-nativepromoter that is functional in plant cells. A plant promoter can be usedas a 5′ regulatory element for modulating expression of an operablylinked gene or genes. Plant promoters may be defined by their temporal,spatial, or developmental expression pattern. The nucleic acid moleculesdescribed herein may comprise nucleic acid sequences comprisingpromoters.

Sequence identity: The similarity between two nucleic acid sequences orbetween two amino acid sequences is expressed in terms of the level ofsequence identity shared between the sequences. Sequence identity istypically expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences. Methods for aligningsequences for comparison are described in detail below.

Analogous position in an amino acid sequence: Nucleic acid and aminoacid sequences may be aligned by the methods described in the followingparagraphs. When aligned, a position in one sequence is in “an analogousposition” with a position in the aligned sequence if the positions areidentical within the consensus sequence.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in: Smith andWaterman, Adv. Appl. math. 2:482, 1981; Needleman and Wunsch, J. Mol.Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237-44, 1988; Higgins andSharp, CABIOS 5:151-3, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; Huang, et al., Computer Applications in theBiosciences 8:155-65, 1992; Pearson et al., Methods in Molecular Biology24:307-31, 1994; Tatiana et al., FEMS Microbiol. Lett., 174:247-50,1990. Altschul et al., J. Mol. Biol. 215:403-10, 1990 (detailedconsideration of sequence-alignment methods and homology calculations).

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST) is available on the Internet (atblast.ncbi.nlm.nih.gov/Blast.cgi), for use in connection withsequence-analysis programs, for example, blastp and blastn. Adescription of how to determine sequence identity using this program isavailable on the Internet through NCBI atblast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs.

For comparisons of amino acid sequences, the “Blast 2 sequences”function of the BLAST program (bl2seq) is employed using the defaultparameters. Specific parameters may be adjusted within the discretion ofone of skill in the art, to for example, provide a penalty for amismatch or reward for a match.

Transformed: As used herein, the term “transformed” refers to a cell,tissue, organ, or organism into which has been introduced a foreignnucleic acid molecule, such as a construct. The introduced nucleic acidmolecule may be integrated into the genomic DNA of the recipient cell,tissue, organ, or organism such that the introduced polynucleotidemolecule is inherited by subsequent progeny. A “transgenic” or“transformed” cell or organism also includes progeny of the cell ororganism and progeny produced from a breeding program employing such atransgenic plant as a parent in, for example, a cross and exhibiting analtered phenotype resulting from the presence of a foreign nucleic acidmolecule.

IV. Systematic Metabolic Engineering Approaches to Accumulating UnusualFatty Acids in a Host Cell, Tissue, or Organism

A. Overview

An embodiment of the invention includes a systematic approach tometabolic engineering the accumulation of ω-7 fatty acids (FA),comprised of palmitoleic (16:1Δ⁹) and vaccenic acid (18:1Δ¹¹), forexample, in plant seeds. To exemplify methods for intercepting the fluxof newly synthesized fatty acids in the plastids, Com25, a 16:0-ACPdesaturase resulting from a directed evolution program to enhance the16:0-desaturase activity of the castor Δ⁹-18:0-desaturase, was expressedunder the control of the seed-specific phaseolin promoter. Anyseed-specific promoter may be used in the embodiments disclosed herein.This approach increased the accumulation of ω-7 FA from less than 2% inwild type (WT) to about 14% in Com25 transformants.

In further exemplary approaches, expression of Com25 in the fab1/fae1double mutant, which is impaired in plastidial and extraplastidial fattyacid elongation, respectively, resulted in increased ω-7 FA accumulationto about 50%. Moreover, introducing an additional Com25 under thecontrol of the LTP170 promoter increased ω-7 FA accumulation to about58%, suggesting that desaturase activity limitation, likely resultingfrom its low turnover rate, had been overcome. The phaseolin:Com25construct was expressed in a series of KASII-deficient backgrounds andω-7 FA content increasing proportionately with 16:0 content up to about30% with total ω-7 FA accumulation up to about 55% was observed.Interestingly, transgenics accumulating 56% ω-7 FA still contained about19% 16:0, more than twice that of WT plants. Expression ofextraplastidial 16:0 desaturases to intercept the flux of 16:0 en routeto triacylglycerol was investigated. Co-expression of plastidial andextraplastidial desaturases along with suppression of KASII in thefab1/fae1 double mutant background resulted in increased accumulation ofω-7 FA from about 2% in WT to about 71% in the best engineered line,equivalent to that found in Doxantha seeds.

ω-7 FAs were selected as the target because their synthesis in naturalsystems, like those of other unusual FA, is initiated by modification ofan intermediate of FAS by a variant desaturase enzyme. Cahoon et al.,(1997) Plant Mol. Biol. 33:1105-10; and Cahoon et al., (1998) PlantPhysiol. 117(2):593-8. In addition, ω-7 FA have potential commercialapplications as polymer feedstocks while having similar physicalproperties to naturally occurring unsaturated fatty acids.

Metabolic engineering studies were initiated by introducing a previouslyunreported Δ⁹-16:0-acyl carrier protein (ACP) desaturase, Com25, intothe model plant Arabidopsis under the control of a seed-specificpromoter. Approaches to diverting carbon flow into ω-7 FA by the choiceof mutant backgrounds that contain elevated levels of 16:0 and by theco-expression of constructs designed to divert the flux of carbon intothe target fatty acid by affecting competition for substrate wereexplored. Extraplastidial desaturase enzymes were expressed todesaturate residual 16:0 after export from the plastid.

Co-expression of plastidial and extraplastidial desaturases along withsuppression of KASII in the fab1/fae1 background resulted in increasedaccumulation of ω-7 FA to up to about 71% from less than 2% in WT,higher than that found in Asclepias and equivalent to that found inDoxantha seeds.

16:0-ACP, the precursor of ω-7 fatty acids, is at the first branch pointof fatty acid biosynthesis, being competed for by the FatB thioesteraseand the KASII elongase; and the introduction of a 16:0-ACP desaturasemakes this a three-way competition. Suppression of KASII and FATB areeffective ways to reduce competition for substrate and increase theaccumulation of ω-7 FA. The increase in ω-7 FA accumulation is saturableat about 30% in the host line, because above this level the desaturaseis limiting. Increasing the dosage of Com25 by expressing a second copyunder the control of a seed-specific promoter further increased theaccumulation of ω-7 fatty acids. However, the seeds of high ω-7 FAaccumulators also contained levels of 16:0 in the range of about 20%,presenting an opportunity to desaturate extraplastidial 16:0. Expressionof two extraplastidial desaturases increased the accumulation of ω-7 FA,resulting in an approximately 50% decrease of 16:0 in mature seeds.

As described in more detail below, systematic metabolic engineering canbe a successful strategy to engineer levels of unusual fatty acidaccumulation comparable to those observed in natural sources because thebest fab1/fae1/Com25/LnΔ9D and AnΔ9D lines accumulate 71% ω-7 FA,substantially higher levels than in Asclepias and equivalent to thelevels found in Doxantha seeds.

B. Nucleic Acids

Nucleic acid sequences in some embodiments of the present invention showincreasing percentage identities when aligned with SEQ ID NO:1. Specificnucleic acid sequences within these and other embodiments may comprisesequences having, for example, at least 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%96%, 97%, 98%, or 100% identity with SEQ ID NO:2. It is understood bythose of ordinary skill in the art that nucleic acid molecules may bemodified without substantially changing the amino acid sequence of anencoded polypeptide, for example, according to permissible nucleotidesubstitutions according to codon degeneracy.

In some embodiments, nucleic acid molecules of the present inventioncomprise promoters. Promoters may be selected on the basis of the celltype into which the vector construct will be inserted. Promoters whichfunction in bacteria, yeast, and plants are well-known in the art. Thepromoters may also be selected on the basis of their regulatoryfeatures. Examples of such features include enhancement oftranscriptional activity, inducibility, tissue-specificity, anddevelopmental stage-specificity. In plants, promoters that areinducible, of viral or synthetic origin, constitutively active,temporally regulated, and spatially regulated have been described (Forexample, see Poszkowski, et al., (1989) EMBO J. 3:2719; Odell et al.,(1985) Nature 313:810; Chau et al., (1989) Science 244:174-81).

Often used constitutive promoters include, for example, the CaMV 35Spromoter, the enhanced CaMV 35S promoter, the Figwort Mosaic Viruspromoter, the mannopine synthase promoter, the nopaline synthasepromoter, and the octopine synthase promoter.

Useful inducible promoters include, for example, promoters induced bysalicylic acid or polyacrylic acids induced by application of safeners(substituted benzenesulfonamide herbicides), heat-shock promoters, anitrate-inducible promoter derived from the spinach nitrate reductasetranscribable nucleic acid molecule sequence, hormone-induciblepromoters, and light-inducible promoters associated with the smallsubunit of RuBP carboxylase and LHCP families.

Examples of useful tissue-specific, developmentally-regulated promotersinclude the β-conglycinin 7Sα promoter and seed-specific promoters.Plant functional promoters useful for preferential expression in seedplastid include those from proteins involved in fatty acid biosynthesisin oilseeds and from plant storage proteins. Examples of such promotersinclude the 5′ regulatory regions from such transcribable nucleic acidmolecule sequences as phaseolin, napin, zein, soybean trypsin inhibitor,ACP, stearoyl-ACP desaturase, and oleosin. Another exemplarytissue-specific promoter is the lectin promoter, which is specific forseed tissue.

Other useful promoters include the nopaline synthase, mannopinesynthase, and octopine synthase promoters, which are carried ontumor-inducing plasmids of Agrobacterium tumefaciens; the cauliflowermosaic virus (CaMV) 19S and 35S promoters; the enhanced CaMV 35Spromoter; the Figwort Mosaic Virus 35S promoter; the light-induciblepromoter from the small subunit of ribulose-1,5-bisphosphate carboxylase(ssRUBISCO); the EIF-4A promoter from tobacco (Mandel et al., (1995)Plant Mol. Biol. 29:995-1004); corn sucrose synthetase; corn alcoholdehydrogenase I; corn light harvesting compolex; corn heat shockprotein; the chitinase promoter from Arabidopsis; the LTP (LipidTransfer Protein) promoters; petunia chalcone isomerase; bean glycinerich protein 1; potato patatin; the ubiquitin promoter; and the actinpromoter. Useful promoters are preferably seed-selective, tissueselective, or inducible. Seed-specific regulation is discussed in, forexample, EP 0 255 378.

C. Amino Acid Sequences

Amino acid sequences according to some embodiments of the presentinvention show increasing percentage identities when aligned with SEQ IDNO:2. Specific amino acid sequences within these and other embodimentsmay comprise sequences having, for example, at least 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%96%, 97%, 98%, or 100% identity with SEQ ID NO:2. In many embodiments,the amino acid sequence having the aforementioned sequence identity whenaligned with SEQ ID NO:2 encodes a peptide with enzymatic Δ⁹-18:0-ACPdesaturase activity.

D. Alteration of Com25: 5 Mutations

Aspects of the present invention concern novel genetically engineereddesaturases derived from a parental castor desaturase. In specificembodiments, the genetically engineered desaturase is Com25. Com25differs from the parental castor desaturase at the following 5 aminoacid positions: M114S, T117R, L118C, P179L, and G188T (numberedaccording to the mature castor desaturase PDB entry 1AFR). In furtherembodiments, the genetically engineered desaturase may comprise one ormore of these 5 mutations in Com25. For example, a geneticallyengineered desaturase may differ from the parental castor desaturase atthe following positions: M114S; T117R; L118C; P179L; G188T; M114S andT117R; M114S and L118C; M114S and P179L; M114S and G188T; T117R andL118C; T117R and P179L; T117R and G188T; L118C and P179L; L118C andG188T; P179L and G188T; M114S, T117R, and L118C; M114S, T117R, andP179L; M114S, T1117R, and G188T; M114S, L118C, and P179L; M114S, L118C,and G188T; M114S, P179L, and G188T; T117R, L118C, and P179L; T117R,L118C, and G188T; T117R, P179L, and G188T; or L118C, P179L, and G188T.

E. Hosts Containing Increased Levels of 16:0 Fatty Acids.

In preferred embodiments, host cells or materials transformed with Com25may exhibit increased levels of 16:0 fatty acids. Host cells may exhibitincreased levels of 16:0 fatty acids, for example, by having metabolismof 16:0-ACP reduced in those host cells. Other methods of increasinglevels of 16:0 fatty acids in a host cell may be used, and such methodsmay be chosen by the exercise of the discretion of one of skill in theart. Examples of methods of increasing levels of 16:0 fatty acids in ahost cell include, but are not limited to: 1) expression of anextraplastidial desaturase in the host cell; 2) suppression of KASII inthe host cell, for example by introducing a mutation in the fab1 gene;and 3) decreasing elongation of 16:0 fatty acids, for example, byintroducing a mutation in the fae1 gene.

F. Methods for Genetic Transformation of Plant Material

The present invention is also directed to methods of producingtransformed cells which comprise one or more nucleic acid moleculescomprising a nucleic acid sequence at least 60% identical to SEQ IDNO:1. Such nucleic acid molecules may also comprise, for example,non-coding regulatory elements, such as promoters. Other sequences mayalso be introduced into the cell along with the non-coding regulatoryelements and transcribable nucleic acid molecule sequences. These othersequences may include 3′ transcriptional terminators, 3′poly-adenylation signals, other untranslated sequences, transit ortargeting sequences, selectable markers, enhancers, and operators.

The method of transformation generally comprises the steps of selectinga suitable host cell, transforming the host cell with a recombinantvector, and obtaining the transformed host cell.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. These methods can generally be classified into fivecategories: (1) chemical methods (Graham and Van der Eb (1973) Virology54(2):536-9; Zatloukal et al., (1992) Ann. N.Y. Acad. Sci. 660:136-53);(2) physical methods such as microinjection (Capechi (1980) Cell22(2):479-88), electroporation (Wong and Neumann, Biochim. Biophys. Res.Commun. (1982) 107(2):584-7; Fromm et al., (1985) Proc. Natl. Acad. Sci.USA 82(17):5824-8; U.S. Pat. No. 5,384,253), and particle acceleration(Johnston and Tang (1994) Methods Cell Biol. 43(A):353-65; Fynan et al.,(1993) Proc. Natl. Acad. Sci. USA 90(24):11478-82; (3) viral vectors(Clapp (1993) Clin. Perinatol. 20(1):155-68; Lu et al., (1993) J. Exp.Med. 178(6):2089-96; Eglitis and Anderson (1988) Biotechniques6(7):608-14); (4) receptor-mediated mechanisms (Curiel et al., (1992)Hum. Gen. Ther. 3(2):147-54; Wagner et al., (1992) Proc. Natl. Acad.Sci. USA 89(13):6099-103); and (5) bacterial-mediated mechanisms, suchas with Agrobacterium. Alternatively, nucleic acids can be directlyintroduced into pollen by directly injecting a plant's reproductiveorgans (Zhou et al., (1983) Methods in Enzymology 101:433; Hess (1987)Intern. Rev. Cytol. 107:367; Luo et al., (1988) Plant Mol. Biol.Reporter 6:165; Pena et al., (1987) Nature 325:274). Othertransformation methods include, for example, protoplast transformationas illustrated in U.S. Pat. No. 5,508,184. The nucleic acid moleculesmay also be injected into immature embryos (Neuhaus et al., (1987)Theor. Appl. Genet. 75:30).

The most commonly used methods for transformation of plant cells are:the Agrobacterium-mediated DNA transfer process (Fraley et al., (1983)Proc. natl. Acad. Sci. USA 80:4803) (as illustrated in U.S. Pat. No.5,824,877; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,981,840; and U.S.Pat. No. 6,384,301) and the biolistics or microprojectilebombardment-mediated process (i.e., the gene gun) (such as described inU.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.6,160,208; Us. Pat. No. 6,399,861; and U.S. Pat. No. 6,403,865).Typically, nuclear transformation is desired, but where it is desirableto specifically transform plastids, such as chloroplasts or amyloplasts,plant plastids may be transformed utilizing a microprojectile-mediateddelivery of the desired nucleic acid molecule for certain plant speciessuch as Arabidopsis, tobacco, potato and Brassica species.

Agrobacterium-mediated transformation is achieved through the use of agenetically engineered soil bacterium belonging to the genusAgrobacterium. Several Agrobacterium species mediate the transfer of aspecific DNA known as “T-DNA,” that can be genetically engineered tocarry any desired piece of DNA into many plant species. The major eventsmarking the process of T-DNA mediated pathogensis are: induction ofvirulence genes, and processing and transfer of TDNA. This process isthe subject of many reviews (Ream (1989) Ann. Rev. Phytopathol.27:583-618; Howard and Citovsky (1990) Bioassays 12:103-8; Kado (1991)Crit. Rev. Plant Sci. 10:1-32; Zambryski (1992) Annual Rev. PlantPhysiol. Plant Mol. Biol. 43:465-90; Gelvin (1993) in Transgenic Plants,Kung and Wu eds., Academic Press, San Diego, pp. 49-87; Binns and Howitz(1994) In Bacterical Pathogenesis of Plants and Animals, Dang, ed.,Berlin: Springer Verlag., pp. 119-38; Hooykaas and Beijersbergen (1994)Ann. Rev. Phytopathol. 32:157-79; Lessl and Lanka (1994) Cell 77:321-4;Zupan and Zambryski (1995) Annual Rev. Phytopathol. 27:583-618).

To select or score for transformed plant cells regardless oftransformation methodology, the DNA introduced into the cell may containa gene that functions in a regenerable plant tissue to produce acompound that confers upon the plant tissue resistance to an otherwisetoxic compound. Genes of interest for use as a selectable, screenable,or scorable marker include, but are not limited to, GUS, greenfluorescent protein (GFP), luciferase, and antibiotic or herbicidetolerance genes. Examples of antibiotic resistance genes include genesconferring resistance to the penicillins, kanamycin (and neomycin, G418,bleomycin); methotrexate (and trimethoprim); chloramphenicol; andtetracycline. For example, glyphosate resistance may be conferred by aherbicide resistance gene. Della-Cioppa et al., (1987) Bio/Technology5:579-84. Other selection devices can also be implemented including butnot limited to tolerance to phosphinothricin, bialaphos, and positiveselection mechanisms, Joersbro et al., (1998) Mol. Breed. 4:111-7, andare considered within the scope of the present invention.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, may then be allowedto mature into plants.

The presently disclosed methods may be used with any transformable plantcell or tissue. Transformable cells and tissues, as used herein,includes but is not limited to those cells or tissues that are capableof further propagation to give rise to a plant. Those of skill in theart recognize that a number of plant cells or tissues are transformablein which after insertion of exogenous DNA and appropriate cultureconditions the plant cells or tissues can form into a differentiatedplant. Tissue suitable for these purposes can include but is not limitedto immature embryos, scutellar tissue, suspension cell cultures,immature inflorescence, shoot meristem, nodal explants, callus tissue,hypocotyl tissue, cotyledons, roots, and leaves.

The regeneration, development, and cultivation of plants fromtransformed plant protoplast or explants is known in the art. Weissbachand Weissbach (1988) Methods for Plant Molecular Biology, (Eds.)Academic Press, Inc., San Diego, Calif.; Horsch et al., (1985) Science227:1229-31. This regeneration and growth process typically includes thesteps of selecting transformed cells and culturing those cells throughthe usual stages of embryonic development through the rooted plantletstage. Transgenic embryos and seeds are similarly regenerated. In thismethod, transformants are generally cultured in the presence of aselective media which selects for the successfully transformed cells andinduces the regeneration of plant shoots. Fraley et al., (1993) Proc.Natl. Acad. Sci. USA 80:4803. These shoots are typically obtained withintwo to four months. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Cells that survive the exposure to a selective agent, or cells that havebeen scored positive in a screening assay, may be cultured in media thatsupports regeneration of plants. The shoots may then be transferred toan appropriate root-inducing medium containing the selective agent andan antibiotic to prevent bacterial growth. Many of the shoots willdevelop roots. These are then transplanted to soil or other media toallow the continued development of roots. The method, as outlined above,will generally vary depending on the particular plant strain employed,and particulars of the methodology are therefore within the discretionof one of skill in the art.

The regenerated transgenic plants may be self-pollinated to providehomozygous transgenic plants. Alternatively, pollen obtained from theregenerated transgenic plants may be crossed with non-transgenic plants,preferably inbred lines of agronomically important species. Conversely,pollen from non-transgenic plants may be used to pollinate theregenerated transgenic plants.

The transgenic plant may pass along the transformed nucleic acidsequence to its progeny. The transgenic plant is preferably homozygousfor the transformed nucleic acid sequence and transmits that sequence toall of its offspring upon, and as a result of, sexual reproduction.Progeny may be grown from seeds produced by the transgenic plant. Theseadditional plants may then be self-pollinated to generate a truebreeding line of plants.

The progeny from these plants may be evaluated, among other things, forgene expression. The gene expression may be detected by several commonmethods such as western blotting, northern blotting,immunoprecipitation, and ELISA (Enzyme-Linked ImmunoSorbent Assay). Thetransformed plants may also be analyzed for the presence of theintroduced DNA and the expression level and/or fatty acid profileconferred by the nucleic acid molecules and amino acid molecules of thepresent invention. Those of skill in the art are aware of the numerousmethods available for the analysis of transformed plants. For example,methods for plant analysis include, but are not limited to, Southernblots or northern blots, PCR-based approaches, biochemical assays,phenotypic screening methods, field evaluations, and immunodiagnosticassays.

Methods for specifically transforming dicots are well-known to thoseskilled in the art. Transformation and plant regeneration using thesemethods have been described for a number of crops including, but notlimited to, members of the genus Arabidopsis, cotton (Gossypiumhirsutum), soybean (Glycine max), peanut (Arachis hypogaea), and membersof the genus Brassica. Methods for transforming dicots, primarily by useof Agrobacterium tumefaciens and obtaining transgenic plants have beenpublished for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135;U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834; U.S. Pat.No. 5,416,011; McCabe, et al., (1988) Biotechnology 6:923; Christou etal., (1988) Plant Physiol. 87:671-4; Brassica (U.S. Pat. No. 5,463,174);peanut (Cheng et al., (1996) Plant Cell Rep. 15:653-7; McKently et al.,(1995) Plant Cell Rep. 14:699-703; papaya; and pea (Grant et al., (1995)Plant Cell Rep. 15:254-8).

Methods for transforming monocots are also well-known in the art.Transformation and plant regeneration using these methods have beendescribed for a number of crops including, but not limited to, barley(Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard grass(Dactylis glomerata); rice (Oryza sativa, including indica and japonicavarieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tallfescue (Festuca arundinacea); turfgrass species (e.g., Agrostisstolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticumaestivum); and alfalfa (Medicago sativa). It is apparent to those ofskill in the art that a number of transformation methodologies can beused and modified for production of stable transgenic plants for anynumber of target crops of interest.

Any plant may be chosen for use in the presently disclosed methods.Preferred plants for modification according to the present inventioninclude Arabidopsis thaliana, borage (Borago spp.), Canola, castor(Ricinus communis), cocoa bean (Theobroma cacao), corn (Zea mays),cotton (Gossypium spp), Crambe spp., Cuphea spp., flax (Linum spp.),Lesquerella and Limnanthes spp., Linola, nasturtium (Tropaeolum spp.),Oenothera spp., olive (Olea spp.), palm (Elaeis spp.), peanut (Arachisspp.), rapeseed, safflower (Carthamus spp.), soybean (Glycine and Sofaspp.), sunflower (Helianthus spp.), tobacco (Nicotiana spp.), Vernoniaspp., wheat (Triticum spp.), barley (Hordeum spp.), rice (Oryza spp.),oat (Avena spp.) sorghum (Sorghum spp.), rye (Secale spp.) or othermembers of the Gramineae.

It is apparent to those of skill in the art that a number oftransformation methodologies can be used and modified for production ofstable transgenic plants from any number of target crops of interest.

G. Transgenic Seeds

In some embodiments of the invention, a transgenic seed comprises apolypeptide comprising an amino acid sequence at least 90% identical toSEQ ID NO:2. In these and other embodiments, the transgenic seedcomprises a nucleic acid sequence at least 60% identical to SEQ ID NO:1.In certain embodiments, the seeds of the present invention exhibitincreased levels of unusual fatty acids, for example, ω-7 fatty acids,such as 16:1Δ⁹ and/or 18:1Δ¹¹. The seeds can be harvested from fertiletransgenic plants and be used to grow progeny generations of transformedplants of this invention, including hybrid plant lines comprising anucleic acid sequence according to this invention and another gene ornucleic acid construct of interest.

Each document, patent, and reference cited herein is herein incorporatedby its entirety.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example I Materials and Methods

Plant Growth and Transformation

Arabidopsis plants were grown in soil under continuous exposure to 300microeinsteins of light (1 microeinstein=1 mol of light) in E7/2™controlled environment growth chambers (Conviron). The plants weretransformed according to Clough and Bent's method, Clough and Bent(1998) Plant J. 16(6):735-43, using Agrobacterium tumefaciens strainGV3101. We identified individual T₁ seeds carrying the transgenes by thefluorescence emitted, Stuitje et al., (2003) Plant Biotechnol. J.1(4):301-9, upon illumination with green light from an X5 LED™flashlight (Inova) in conjunction with a 25A red camera filter.Pidkowich et al., (2007) Proc. Natl. Acad. Sci. USA 104(11): 4742-7. AWILD™ M3Z dissection microscope equipped with an Olympus U-LH100HG™illumination system was used to discriminate between seeds carryingZs-Green and Ds-Red markers with the use of filters FITC 535 and FITC515, respectively. Seed-specific expression was achieved by placingconstructs under the control of the phaseolin seed-storage proteinpromoter or the LTP170 promoter. Slightom et al., (1983) Proc. Natl.Acad. Sci. USA 80(7):1897-1901; and van der Geest and Hall (1997) PlantMol Biol. 33(3):553-7.

Source of Com25

Com25 is a variant of the Ricinus communis Δ ⁹-18:0-ACP desaturase thatarose from a program of combinatorial saturation mutagenesis/selectiondesigned to identify variants with improved activity towards acyl chainsof less than 18C in lengths. Whittle and Shanklin (2001) J. Biol. Chem.276(24):21500-5. Com25 differs from the parental castor desaturase atthe following 5 amino acid positions: M114S, T117R, L118C, P179L, andG188T (numbered according to the mature castor desaturase PDB entry1AFR).

Plasmid Constructs

Phas:Com25. The entire open reading frame of the castor variant Com25,engineered to contain its authentic transit peptide and flanked by 5′PacI and 3′ XhoI restriction sites was cloned into the correspondingsites of plasmid pDs-Red-Phas, Pidkowich et al., (2007) Proc. Natl.Acad. Sci. USA 104(11): 4742-7, (with Ds-Red marker) to createPhas:Com25 (FIG. 5).

Phas:Com25, LTP170:Com25. The LTP170 promoter was amplified fromArabidopsis genomic DNA using primers P17-5′BamHI(GGGATCCCCGGGTTGACATTTTTACCTTTTT; SEQ ID NO:3) and P17-3′PacI(GGTTAATTAAGTCTTCAAACTCTAGGA; SEQ ID NO:4), subcloned into pGEMT-Easybefore isolation of the BamHI-PacI fragment, which was cloned into thecorresponding sites of plasmid pDs-Red-Phas:Com25 (described, supra) tocreate pDs-Red-LTP170:Com25. A fragment containing Com25 along with thephaseolin terminator was excised using BamHI and EcoRV and cloned intothe BamHI and SmaI restriction sites within vector pDs-Red-LTP170-Com25to create Phas:Com25/LTP170:Com25 (FIG. 5).

Phas:Fab1-HPAS. This construct was created in two steps; first theconstruction of Phas:FatB-HP, and afterwards the insertion of anantisense portion of the FatB gene to replace part of the Fad2 intronseparating the sense and antisense portions of the FatB gene comprisingthe hairpin. To achieve this, 150 bp of the Arabidopsis FatB 3′UTR wasamplified from genomic DNA in both sense (using primers FatB-hps-5′PstIGGGCTGCAGAAACAAGTTTCGGCCACCAACCC; SEQ ID NO:5 and FatB-hps-3′XhoICCCCTCGAGACATCAGAATTCGTAATGAT; SEQ ID NO:6) and antisense (using primersFatB-hpa-5′NheI GGGGCTAGCAAGTTTCGGCCACCAACCC; SEQ ID NO:7 andFatB-hpa-3′PacI CCCTTAATTAAACATCAGAATTCGTAATGAT; SEQ ID NO:8)orientations. These fragments were restricted with PstI/XhoI andNheI/PacI and used to replace the 5′UTR sense and antisense portions ofFabI in pGEM-T-Easy-HTM3, Pidkowich et al., (2007) Proc. Natl. Acad.Sci. USA 104(11): 4742-7, at their equivalent sites, to create theintermediate plasmid pGEM-T-Easy-HTM4. To create a 300 bp antisenseportion of the FatB coding region, a fragment was amplified with primersFatB-Exon-5′Sp-Bam (CCACTAGTGGATCCACCTCTGCTACGTCGTCATT; SEQ ID NO:9) andFatB-Exon-3′Bg-Sal (GGAGATCTGTCGACGTAGTTATAGCAGCAAGA AG; SEQ ID NO:10),and the fragment, restricted with BamHI and SalI, was used to replacepart of the Fad2-intron after restriction with BglII and SpeI to createpGEM-T-Easy-HTM5.

The assembled HPAS fragment was excised with the use of PacI and XhoIand cloned into the equivalent sites of pZs-Green-Phas:Com25 (plasmidpDs-Red-Phas:Com25, described, supra, in which the fluorescence markerpCVMV:Ds-Red had been replaced by a green fluorescent protein markerpCVMV:Zs-Green™ (Clonetech)) to create plasmid Phas:FatB-HPAS (FIG. 5).

Phas:AnD9d, Phas:LnD9D. Two fungal acyl-CoA D9 desaturases were combinedin plasmid pDAB7318 with both genes being driven by the seed-specificPhas promoter from Phaseolus vulgaris. The first gene in the constructwas an acyl-CoA Δ9-desaturase from Aspergillus nidulans that wasredesigned and synthesized for optimal expression in plants (US PatentApplication 20080260933A1) and fused to the 3′ untranslated region and3′ MAR from the Phaseolus vulgaris phaseolin gene. The second desaturasegene in this construct was an acyl-CoA A9-desaturase from Leptosphaerianodorum that was also redesigned and synthesized for plant expressionand fused to the Agrobacterium tumefaciens ORF23 3′ untranslated region.This desaturase was identified by homology searches of the S. nodorumgenome sequence released by the Leptosphaeria nodorum SequencingProject, Broad Institute of Harvard and MET (http://www.broad.mit.edu).It was shown to have a preference for desaturation of palmitate bycomplementation of the oleI mutant of Saccharomyces cerevisiae.Phas:Fab1-HPAS-Phas:Com25. To simplify gene stacking experiments,plasmid Phas:Fab1/HPAS-Phas:Com25 was constructed to combine Com25expression with KASII suppression. To achieve this, the Phaseolinterminator was isolated from Phas:Com25 and cloned into the intermediatevector pBL, with the EcoRV-EcoRV fragment containing the Phaseolinpromoter driving Com25, to create pBL-Phas:Com25-PhasTer. This Com25expression cassette was excised using flanking EcoRI-EcoRI restrictionsites and cloned into the corresponding site within Phas:Fab1-HPAS tocreate Phas:Fab1-HPAS-Phas-Com25. See FIG. 5.

Fatty-Acid Analysis

To analyze the fatty acids of single seeds, fatty-acid methyl esters(FAMEs) were prepared by incubating the seeds with 0.2Mtrimethylsulfonium hydroxide in methanol. Butte et al., (1982) Anal.Lett. 15(10):841-50. To similarly analyze bulk seeds, FAMEs wereprepared by incubating the seeds in 0.5 mL BCl₃ for 1 h at 80° C.,extracting them with 1 mL hexane, and then drying under N₂. FAMEs wereanalyzed either with an HP6890™ gas chromatograph-flame ionizationdetector (Agilent Technologies), or an HP5890™ gas chromatograph-massspectrometer (Hewlett-Packard) fitted with 60-m×250-μM SP-2390 capillarycolumns (Supelco). The oven temperature was raised during the analysesfrom 100° C. to 240° C. at a rate of 15° C./min with a flow rate of 1.1mL/min. Mass spectrometry was performed with an HP5973™ mass selectivedetector (Hewlett-Packard). We determined the double-bond positions ofmonounsaturated FAMEs by dimethyl sulfide derivatization. Yamamoto etal., (1991) Chem. and Phys. Lipids 60(1):39-50.

Example II Expression of Com25 in WT Arabidopsis

Several plants including Asclepias, Hopkins and Chisholm (1961) Can. J.Biochem. Physiol. 39:829-35, and Doxantha, Chisholm and Hopkins (1965)J. Am. Oil Chem. Soc. 42:49-50, have been reported to accumulate ω-7 FAin their seeds. Genes encoding the desaturase enzymes responsible forsynthesizing palmitoleate have been isolated. The activities of thecorresponding recombinant desaturase enzymes, Cahoon et al., (1997)Plant Mol. Biol. 33:1105-10; Cahoon et al., (1998) Plant Physiol.117(2):593-8, were, like those for many variant desaturases, lower thanthose reported for archetypal stearoyl-ACP desaturases. Whittle andShanklin (2001) J. Biol. Chem. 276(24):21500-5. We compared the effectsof expressing the Asclepias and Doxantha desaturases and severalvariants of the castor desaturase, including the desaturase from castorvariety 5.2 and Com25 that arose from directed evolution experimentsdesigned to enhance the 16:0-desaturase activity of the castorΔ⁹-18:0-desaturase, Whittle and Shanklin (2001) J. Biol. Chem.276(24):21500-5, in Arabidopsis. In these experiments, Com25outperformed the other desaturase enzymes, Bondaruk et al., (2007) PlantBreeding 126:186-94, increasing the accumulation of 16:1Δ⁹ and itselongation product, 18:1Δ¹¹ in WT Arabidopsis from barely detectablelevels in untransformed plants to about 2% and about 12% respectively;yielding a total of about 14% ω-7 fatty acids in Com25 transformants.FIG. 2A; FIG. 2B.

Table 1 shows that while Com25 has a much improved k_(cat) (11.1 min⁻¹)over castor WT (2.8 min⁻¹) for 16:0-ACP substrate, it falls short ofthat reported for castor variant 5.2 (25.3 min-1). Whittle and Shanklin(2001) J. Biol. Chem. 276(24):21500-5. However, Com25's K_(m) for16:0-ACP (0.12 μM) is 4.6-times lower than that of castor variant 5.2(0.55) and 42-times lower than that of castor WT (5.0). The resultingspecificity factor with 16:0-ACP substrate for com25 of 91 μM⁻¹ min⁻¹ isapproximately twice that of castor variant 5.2 and 163-times that ofcastor WT. Indeed, Com25's specificity factor with 16:0-ACP isequivalent to that of castor WT with its natural 18:0-ACP substrate (92μM⁻¹·min⁻¹). Com25's improved K_(m) for 16:0-ACP relative to castorvariant 5.2 suggests it completes more effectively with FatB and KASIIfor substrate, providing an explanation as to why its expressionfacilitates greater accumulation of ω-7 FA than castor variant 5.2,despite its lower k_(cat).

Kinetic parameters of castor desaturase and its variants with varioussubstrates.

TABLE 1 Enzyme Substrate k_(cat) specificity factor min^(−1b) μM μM⁻¹ ·min⁻¹ K_(m) k_(cat)/K_(m) com25 16:0-ACP 11.1 (0.6) 0.12 (0.03) 91 5.216:0-ACP 25.3 (1.1) 0.55 (.06)  46 WT 16:0-ACP  2.8 (0.1) 5.0 (0.5) 0.56WT 18:0-ACP 42.3 (1.6) 0.46 (0.05) 92

Example IV Expression of Com25 in Hosts Containing Increased Levels of16:0

In WT Arabidopsis, fatty acids are synthesized de novo via the ACP trackto a first branch point at the level of 16:0-ACP. FIG. 1. If acted on byFATB, the palmitoyl thioesterase, 16:0 free fatty acid is released fromthe plastid to the cytoplasm where it is esterified to CoA, andsubsequently transesterified onto phospholipids of the endomembranesystem. Alternatively, β-ketoacyl-ACP synthase II (KASII) elongates themajority of 16:0-ACP to 18:0-ACP whereupon it is desaturated by theΔ⁹-stearoyl-ACP desaturase to produce oleoyl-ACP. FATA, the oleoyl-ACPthioesterase releases the oleic acid, which exits the plastid and, likethe palmitate, becomes activated to the CoA-thioester and is transferredto the phospholipids. In the ER, oleate can be elongated to 10:1Δ¹¹ viathe action of the fatty acid elongase (FAE) I, or become sequentiallydesaturated by the action of FAD2 and FAD3 to produce linolenic orlinolenic acids respectively.

16:0-ACP is the earliest metabolite in the FA synthesis pathway that canbe committed to ω-7 production by its desaturation to 16:1Δ⁹-ACP. Toachieve this, the feasibility of expressing a plastidialΔ⁹-16:0-ACP-specific desaturase under the control of a seed-specificpromoter was explored (see FIG. 1 (reaction 1)).

As described, supra, β-ketoacyl-ACP synthase II (KAS II) elongates16:0-ACP to 18:0-ACP. Therefore, lines with lowered KASII activity weresought that would contain increased levels of 16:0 substrate. FIG. 2shows representative GC traces of seed FA methyl esters. Despite manymutagenesis screens, only one mutant fab1 has been reported, James andDooner (1990) Theor. Apple Genet. 80:241-45, that exhibits increased16:0 levels in leaves and seeds, which contain about 21% of 16:0compared to about 10% in WT, as shown in FIG. 2C; and Table 2.Biochemical evidence showed that the fab1 lesion is in KASII, becauseits activity was reduced in the mutant. Carlsson et al., (2002) Plant J.29(6):761-70. Expression of Com25 in fab1 increased the accumulation of16:1Δ⁹ and 18:1Δ¹¹ to about 23% and about 16% respectively, yielding atotal of about 39% ω-7 FA. FIG. 2D; and Table 2. This large increase ofω-7 FA upon the expression of Com25 in the fab1-1 background correlateswith the increase in total 16:0 accumulation in mature seeds and likelyresults from decreased competition for 16:0-ACP substrate by KASII.

We also combined the fab1 mutation with fae1, because its deficiency inextraplastidial elongation of C18 to C20 fatty acids further increasesthe amount of 16:0 fatty acids, and simplifies analysis. Theuntransformed double mutant contains about 9% of ω-7 fatty acids,presumably reflecting increased desaturation of 16:0-ACP by theΔ⁹-18:0-ACP desaturase in the presence of increased levels of 16:0-ACPsubstrate. FIG. 2E; and Table 2. Expression of Com25 in fab1/fae1resulted in an increase of 16:1Δ⁹ and 18:1Δ¹¹ to about 26% and about 23%respectively, yielding an increase of ω-7 FA to about 50%. FIG. 2F; andTable 2.

From the above results, increased accumulation of 16:1 in fab1 andfab1/fae1 seeds correlates with increased 16:0, and so we sought linesto express Com25 in which 16:0 levels were higher than that of thefab1/fae1 double mutant. Two such mutants were recently reported inwhich Fab1 was suppressed, one by hairpin (HP)RNAi, Pidkowich et al.,(2007) Proc. Natl. Acad. Sci. USA 104(11): 4742-7, and the other by anovel method of suppression termed hairpin-antisense (HPAS)RNAi, Nguyenand Shanldin (2009) Journal of the American Oil Chemists Society86:41-9. These lines contain strongly elevated seed 16:0 accumulationlevels at 42% and 46%, respectively. FIG. 3. Transformation with Com25yielded a further increase of about 5% of ω-7 FA in both cases. Table 2.Thus, increases in 16:0 accumulation at low levels of 16:0 is predictiveof increased Com25 desaturation as evidenced by proportional increasesin ω-7 FA accumulation, but this response is apparently saturable at alittle over 30%, as there was no difference between ω-7 FA accumulationexpression in hosts accumulating either 42% or 46% 16:0. FIG. 3. Withoutintending to be bound by any particular theory, it is possible thatfactors other than substrate, i.e., desaturase abundance and/oravailability of reductant become limiting in these transgenics.

Recently, a T-DNA knockout allele, fab1-2 was described that hasincreased 16:0 levels in the heterozygote; however, the homozygote wasshown to be embryo lethal. Pidkowich et al., (2007) Proc. Natl. Acad.Sci. USA 104(11): 4742-7. We hypothesized that the lethal phenotyperesults from the reduction of unsaturated fatty acids, and theorize thatexpression of Com25 in this line may confer viability. In contrast, thefab1/fae1 double mutant is viable in the homozygous condition and isindistinguishable in growth and development from WT. We therefore usedthe fab1/fae1 double mutant as an experimental host for subsequentexperiments.

Example V Increasing Com25 Gene Dosage Results in Increased ω-7Accumulation

The archetypal castor Δ⁹-18:0-ACP desaturase has a k_(cat) of 42 min⁻¹,Whittle and Shanklin (2001) J. Biol. Chem. 276(24):21500-5; which isseveral-fold higher than those reported for Δ⁹-16:0-ACP desaturases.Cahoon et al., (1997) Plant Mol. Biol. 33:1105-10; and Cahoon et al.,(1998) Plant Physiol. 117(2):593-8. While this turnover is comparable tothose of similar Fe-dependent oxidation reactions such as cytochromeP450s, these rates are lower (in many cases, by orders of magnitude)than many metabolic enzymes. The low turnover rates of desaturasesnecessitate high levels of protein expression in order to account forthe desaturation of a large proportion of the carbon stored in the seed,raising the possibility that the abundance of desaturase enzyme mightlimit ω-7 accumulation. To test this hypothesis, Com25 was engineeredunder the control of a seed-specific LTP170 promoter (that controls theexpression of a seed storage protein) and co-expressed this along withthe phaseolin-driven Com25 construct described above. Co-expression ofCom25 under the control of the phaseolin and LTP170 promoters in thefab1/fae1 background resulted in an increase of ω-7 FA accumulation fromabout 50% to about 58%; the increase in the 16:1Δ⁹ being larger (about6%) than that of 18:1Δ¹¹ (about 2%). This increase in ω-7 FAaccumulation is moderate suggesting that Com25 is likely not limiting inseeds expressing both Com25 constructs.

TABLE 2 % Fatty acid Plant 16:0 16:1Δ9 16:2 18:0 18:1Δ9 18:1Δ11 WT 10.4± 0.4  0.1 ± 0.06 0 3.7 ± 0.4 14.5 ± 2.2   1.7 ± 0.2 fab1 20.8 ± 1.6 1.5 ± 0.5 0 3.7 ± 0.4 18.4 ± 1.3   2.8 ± 0.9 fab1, fae1 26.8 ± 1.9  1.9± 0.3 0 3.6 ± 0.5 15.8 ± 1.5   6.8 ± 0.8 WT,  9.2 ± 1.5  1.6 ± 0.4 0 3.7± 0.3 5.7 ± 1.6 12.8 ± 1.2 Com25 fab1, 18.6 ± 2.1 23.5 ± 3.7 1.6 ± 0.51.9 ± 0.3 2.9 ± 2.2 15.6 ± 3.3 Com25 fab1, fae1,   22 ± 2.6 26.2 ± 2.92.0 ± 0.4 1.8 ± 0.4 3.7 ± 1.1 23.4 ± 2.3 Com25 fab1, fae1, 20.7 ± 2.130.3 ± 1.6 2.2 ± 0.2 0.9 ± 0.6 4.7 ± 1.4 24.5 ± 1.8 com25 Fab1-HPASfab1, fae1 20.8 ± 0.8 32.5 ± 1.7 2.5 ± 0.4 0.5 ± 0.2 1.9 ± 0.7 25.5 ±1.2 Com25, Com25 fab1, fae1, 23.1 ± 1.5  4.2 ± 0.6 0 2.9 ± 0.8 36.1 ±3.9   5.1 ± 1.7 FatB-HPAS fab1, fae1, 19.1 ± 1.3 26.7 ± 2.1 0 1.9 ± 0.54.8 ± 1.1 28.9 ± 2.3 FatB-HPAS Com25 fab1, fae1 12.7 ± 2.1 17.9 ± 1.80.8 ± 0.1 0.2 ± 0.1 17.7 ± 0.9   5.8 ± 0.7 AnΔ9D, LnΔ9D fab1, fae1,Fab1-HPAS 11.2 ± 1.3 43.4 ± 3.3 0.7 ± 0.5 0.9 ± 0.2 4.6 ± 1.5 23.2 ± 1.1Com25, AnΔ9D, LnΔ9D Doxantha 18.0 ± 0.5 54.6 ± 1.7 2.4 ± 0.5 2.0 ± 0.11.8 ± 0.2 17.3 ± 1.5 % Fatty acid Plant 18:2 18:3 20:0 20:1 Tot ω-7 Δ WT26.6 ± 0.9  20.2 ± 1.1 2.1 ± 0.3 20.7 ± 0.6  1.8 ± 0.2 — fab1 14.8 ±2.7  19.1 ± 1.3 3.1 ± 0.8 15.8 ± 1.0  4.3 ± 1.3 2 fab1, fae1 25.3 ± 2.5 19.7 ± 1.5 0.2 ± 0.1 0  8.7 ± 1.1 4 WT, 26.6 ± 1.6  29.6 ± 2.2 2.1 ± 0.4 8.7 ± 2.1 14.4 ± 1.5 12 Com25 fab1, 9.6 ± 1.7 17.1 ± 1.8 1.0 ± 0.4  8.2± 3.4 39.1 ± 1.9 35 Com25 fab1, fae1, 8.6 ± 0.7 12.4 ± 1.8 0 0 49.6 ±1.1 41 Com25 fab1, fae1, 6.6 ± 0.9   10 ± 1.6 0 0 54.8 ± 1.5 5 com25Fab1-HPAS fab1, fae1 5.7 ± 1.7   11 ± 1.3 0 0   58 ± 1.3 8 Com25, Com25fab1, fae1, 14.8 ± 1.5  13.9 ± 2.3 0 0  9.3 ± 2.0 1 FatB-HPAS fab1,fae1, 7.8 ± 1.6 10.8 ± 2.4 0 0 55.6 ± 1.8 6 FatB-HPAS Com25 fab1, fae124.1 ± 1.2  20.8 ± 0.9 0 0 23.7 ± 1.9 15 AnΔ9D, LnΔ9D fab1, fae1,Fab1-HPAS 8.4 ± 1.2  7.6 ± 1.6 0 0 66.6 ± 3.9 12 Com25, AnΔ9D, LnΔ9DDoxantha 3.9 ± 0.7 0 0 0 71.9 ± 1.3 n/a

Example VI Expression of Extraplastidial A9-16:0 Desaturases Increasesω-7 FA Accumulation

As previously discussed, the use of background Arabidopsis thataccumulates high levels of 16:0 correlates with the formation of ω-7 FAupon expression of a 16:0-ACP desaturase, but much of the 16:0 stillleaves the plastid and accumulates in the seed oil. See Table 2.Therefore, two approaches to reduce the accumulation of this 16:0 inseed oil were considered. One strategy was to reduce the activity of thepalmitate thioesterase FATB (FIG. 1) that cleaves 16:0 from 16:0-ACP.Suppression of FATB via HPAS-RNAi reduced 16:0 accumulation by about 3%,with about a 6% increase of the ω-7 FA. See Table 2. The feasibility offurther reducing the accumulation of seed 16:0 beyond what was observedby suppression of FATB by desaturating the 16:0 after export from theplastid was explored.

Free fatty acids released from the plastid become esterified to CoA byacyl Co-A synthases en route to accumulation as triacylglycerols.Shockey et al., (2003) Plant Physiol. 132(2):1065-76. These cytoplasmicfatty acyl Co—As and phospholipid-linked FA represent pools ofsubstrates potentially available for extraplastidial desaturases. Theexpression of extraplastidial fungal Aspergillus nidulans (An) andLeptosphaeria nodurum (Ln) desaturases, either alone or in combination,were evaluated with respect to reducing 16:0 levels in Arabidopsis.Co-expression of two desaturases from Ln and An under the control of thephaseolin promoter yielded promising results in reducing 16:0 in WTArabidopsis. Therefore, the expression of the Ln and An construct alongwith the expression of a single copy of Com25 in a KASII HPAS-RNAisuppression line was tested. Expression of LnΔ9D and AnΔ9 desaturasesresulted in the conversion of approximately half of the 16:0 to 16:1Δ⁹,resulting in a decrease in 16:0 accumulation in seeds from about 19% toabout 11% (approximately the level seen in WT seeds), with acorresponding increase in 16:1Δ⁹ from about 27% to 43%. Levels of18:1Δ¹¹ remained the same in the host fab1/fae1/Com25 line, and thisline transformed with LnΔ9D and AnΔ9 desaturases (about 25% and about23%, respectively), which shows that the fae1 mutant is almost entirelydevoid in 16:1Δ⁹ elongation activity. This strategy of co-expressingplastidial and extraplastidial desaturases yielded a mean accumulationof about 67% ω-7 FA, with individual plants showing greater than 71%.

What is claimed is:
 1. A method for producing a transgenic plantmaterial, the method comprising: introducing a nucleic acid moleculeinto a plant material comprising an LnΔ9D or AnΔ9 desaturase, whereinthe nucleic acid molecule comprises a polynucleotide that is at least60% identical to SEQ ID NO:1 and encodes a plastidial delta-9 desaturasehaving at least 90% identity to SEQ ID NO:2.
 2. The method of claim 1,wherein introducing the nucleic acid molecule comprises transforming theplant material with the nucleic acid molecule.
 3. The method of claim 1,wherein the plant material comprises a means for increasing levels of16:0-ACP in the plant material.
 4. The method of claim 3, wherein themeans for increasing levels of 16:0-ACP in the plant material issuppression of KASII.
 5. The method of claim 4, wherein suppression ofKASII is accomplished by introducing a mutation in the fab1 gene.
 6. Themethod of claim 3, wherein the means for increasing levels of 16:0-ACPin the plant material is decreasing the elongation of 16:0 fatty acidsin the plant material.
 7. The method of claim 6, wherein decreasing theelongation of 16:0 fatty acids in the plant material is accomplished byintroducing a mutation in the fae1 gene.
 8. The method of claim 1,wherein the plant material is obtained from a plant selected from agenus selected from the group comprising Arabidopsis, Borago, Canola,Ricinus, Theobroma, Zea), Gossypium, Crambe, Cuphea, Linum, Lesquerella,Limnanthes, Linola, Tropaeolum, Oenothera, Olea, Elaeis, Arachis,rapeseed, Carthamus, Glycine, Soja, Helianthus, Nicotiana, Vernonia,Triticum, Hordeum, Oryza, Avena, Sorghum, Secale, or other members ofthe Gramineae.
 9. The method of claim 1, wherein the plant materialcomprises two means for increasing levels of 16:0-ACP in the plantmaterial.
 10. The method of claim 9, wherein the first means forincreasing levels of 16:0-ACP in the plant material is suppression ofKASII, and wherein the second means for increasing levels of 16:0-ACP inthe plant material is decreasing the elongation of 16:0 fatty acids inthe plant material.
 11. The method of claim 1, wherein thepolynucleotide encodes a polypeptide comprising a serine at the positionanalogous to position 114 in SEQ ID NO:2; an arginine at the positionanalogous to position 117 in SEQ ID NO:2; a cysteine at the positionanalogous to position 118 in SEQ ID NO:2, a leucine at the positionanalogous to position 179 in SEQ ID NO:2; or a threonine at the positionanalogous to position 188 in SEQ ID NO:2.
 12. The method of claim 1,wherein the polynucleotide encodes a polypeptide comprising a serine atthe position analogous to position 114 in SEQ ID NO:2; an arginine atthe position analogous to position 117 in SEQ ID NO:2; a cysteine at theposition analogous to position 118 in SEQ ID NO:2, a leucine at theposition analogous to position 179 in SEQ ID NO:2; and a threonine atthe position analogous to position 188 in SEQ ID NO:2.
 13. The method ofclaim 1, wherein the polynucleotide encodes a polypeptide having atleast 95% identity to SEQ ID NO:2.
 14. The method of claim 1, whereinthe polynucleotide encodes a polypeptide having at least 98% identity toSEQ ID NO:2.
 15. The method of claim 1, wherein the polynucleotideencodes the polypeptide of SEQ ID NO:2.
 16. The method of claim 1,wherein the plant material is a plant.
 17. A transgenic plant materialcomprising: a polynucleotide at least 60% identical to SEQ ID NO:1,wherein the polynucleotide encodes a plastidial delta-9 desaturase atleast 90% identical to SEQ ID NO:2; and an LnΔ9D or AnΔ9 desaturase. 18.The transgenic plant material of claim 17, wherein the plant materialcomprises a means for increasing levels of 16:0-ACP in the plantmaterial.
 19. The transgenic plant material of claim 18, wherein themeans for increasing levels of 16:0-ACP in the plant material issuppression of KASII.
 20. The transgenic plant material of claim 18,wherein the means for increasing levels of 16:0-ACP in the plantmaterial is decreasing the elongation of 16:0 fatty acids in the plantmaterial.
 21. The transgenic plant material of claim 17, wherein theplant material is obtained from a plant selected from a genus selectedfrom the group comprising Arabidopsis, Borago, Canola, Ricinus,Theobroma, Zea), Gossypium, Crambe, Cuphea, Linum, Lesquerella,Limnanthes, Linola, Tropaeolum, Oenothera, Olea, Elaeis, Arachis,rapeseed, Carthamus, Glycine, Soja, Helianthus, Nicotiana, Vernonia,Triticum, Hordeum, Oryza, Avena, Sorghum, Secale, or other members ofthe Gramineae.
 22. The transgenic plant material of claim 17, whereinthe plant material comprises a mutant fab1 gene or a mutant fae1 gene.23. The transgenic plant material of claim 17, wherein the plantmaterial comprises a mutant fab1 gene and a mutant fae1 gene.
 24. Thetransgenic plant material of claim 17, wherein the plant material is aplant or a seed.
 25. A method for producing a transgenic plant material,the method comprising introducing into a plant material: a nucleic acidmolecule comprising a polynucleotide that is at least 60% identical toSEQ ID NO:1 and encodes a plastidial delta-9 desaturase having at least90% identity to SEQ ID NO:2; and a nucleic acid molecule encoding anLnΔ9D or AnΔ9 desaturase.
 26. The method of claim 25, wherein the plantmaterial comprises a means for increasing levels of 16:0-ACP in theplant material.
 27. The method of claim 26, wherein the means forincreasing levels of 16:0-ACP in the plant material is suppression ofKASII.
 28. The method of claim 26, wherein the means for increasinglevels of 16:0-ACP in the plant material is decreasing the elongation of16:0 fatty acids in the plant material.
 29. The method of claim 25,wherein the plant material is obtained from a plant selected from agenus selected from the group comprising Arabidopsis, Borago, Canola,Ricinus, Theobroma, Zea), Gossypium, Crambe, Cuphea, Linum, Lesquerella,Limnanthes, Linola, Tropaeolum, Oenothera, Olea, Elaeis, Arachis,rapeseed, Carthamus, Glycine, Soja, Helianthus, Nicotiana, Vernonia,Triticum, Hordeum, Oryza, Avena, Sorghum, Secale, or other members ofthe Gramineae.
 30. The method of claim 25, wherein the plant materialcomprises a mutant fab1 gene or a mutant fae1 gene.
 31. The method ofclaim 25, wherein the plant material comprises a mutant fab1 gene and amutant fae1 gene.
 32. The method of claim 25, wherein the plant materialis a plant or a seed.